Compositions and methods for altering the biodistribution of biological agents

ABSTRACT

The invention relates to a new and improved pharmaceutical composition and method for delivery of therapeutic agents. The methods and composition of the invention can be used with several therapeutic agents and can achieve site specific delivery of a therapeutic or diagnostic substance. This can allow for lower doses and for improved efficacy with drugs which traditionally reach targeted sites and can result in improved utility for agents such as oligonucleotides and polynucleotides which are plagued with problems with biodistribution.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of copendingapplication Ser. No. 08/670,999, filed Jun. 28, 1996, the disclosure ofwhich is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to a new and improved pharmaceuticalcomposition and method for delivery of bioactive substances. The methodsand composition of the invention can be used with several agents and canachieve site specific delivery of a biologically active substances. Thiscan allow for lower doses and for improved efficacy with drugsparticularly agents such as oligonucleotides which are plagued withproblems in achieving therapeutic concentrations at targeted organs.

BACKGROUND OF THE INVENTION

[0003] Drug delivery techniques are employed in the formulation of alldrug therapy to augment drug availability, to reduce drug dose, andconsequently to reduce drug-induced side effects. These techniques serveto control, regulate, and target the release of drugs in the body. Thegoals have been to provide less frequent drug administration, tomaintain constant and continuous therapeutic levels of a drug in thesystemic circulation or at a specific target organ site, to achieve areduction in undesirable side effects, and to promote a reduction in theamount and dose concentration required to realize the desiredtherapeutic benefit.

[0004] To date, drug delivery systems have included drug carriers basedupon proteins, polysaccharides, synthetic polymers, erythrocytes, DNAand liposomes. New generation biologicals such as monoclonal antibodies,gene therapy vectors, anti-cancer drugs such as Taxol, viral baseddrugs, and oligo and poly nucleotides have presented several problemswith regard to delivery. In fact drug delivery may be the primary hurdleto achieving mainstream therapeutic use of these biologics whose initialpotential seemed unlimited but whose therapeutic parameters haveprevented realization of full benefit.

[0005] Synthetic oligodeoxyribonucleotides which are chemically modifiedto confer nuclease resistance represent a fundamentally differentapproach to drug therapy. The most common applications to date areantisense oligos with sequences complementary to a specific targetedmRNA sequence. An antisense oligonucleotide approach to therapy involvesa remarkably simple and specific drug design concept in which the oligocauses a mechanistic intervention in the processes of translation or anearlier processing event. The advantage of this approach is thepotential for gene-specific actions which should be reflected in arelatively low dose and minimal non-targeted side effects.

[0006] Phosphorothioate analogs of polynucleotides have chiralinternucleoside linkages in which one df the non-bridging ligands issulfur. The phosphorothioate analog is currently the most commonlyemployed analogue in biological studies including both in vitro and invivo. The most apparent disadvantage of phosphorothioateoligonucleotides include the high cost of preparation of sufficientamounts of high quality material and non-specific binding to proteins.Hence, the primary advantage of antisense approach (low dose and minimalside effects) fall short of expectations.

[0007] Drug delivery efforts with regard to oligonucleotides andpolynucleotides have focused on two key challenges; transfection ofoligonucleotides into cells and alteration of distribution ofoligonucleotides in vivo.

[0008] Transfection involves the enhancement of in vitro cellularuptake. Biological approaches to improve uptake have included viralvectors such as reconstituted viruses and pseudo virions, and chemicalssuch as liposomes. Methods to improve biodistribution have focused onsuch things as cationic lipids, which are postulated to increasecellular uptake of drugs due to the positively charged lipid attractionto the negatively charged surfaces of most cells.

[0009] Lipofection and DC-cholesterol liposomes have been reported toenhance gene transfer into vascular cells in vivo when administered bycatheter. Cationic lipid DNA complexes have also been reported to resultin effective gene transfer into mouse lungs after intratrachealadministration.

[0010] Cationic liposomal delivery of oligonucleotides has also beenaccomplished however, altered distribution to the lung and liver wasexperienced. Asialoglycoprotein poly(L)-lysine complexes have met withlimited success as well as complexation with Sendai virus coat proteincontaining liposomes. Toxicity and biodistribution, however, haveremained significant issues.

[0011] From the foregoing it can be seen that a targeted drug deliverysystem for delivery of biologics, particularly poly and oligonucleotides is needed for these drugs to achieve their fullestpotential.

[0012] One object of this invention is to provide a novel composition ofmatter to deliver a pharmaceutical agent to a targeted site in vivo.

[0013] Another object of the invention is to provide a method fordelivering a pharmaceutical agent, increasing drug bioavailability anddecreasing toxicity.

[0014] Another object of the invention is to provide a method of forminga novel composition of matter for delivering a pharmaceutical agent to atargeted site in vivo by sonicating perfluorocarbon containingmicrobubbles in a nitrogen-free environment.

[0015] Other objects of the inventions will become apparent from thedescription of the invention which follows.

SUMMARY OF THE INVENTION

[0016] According to the invention a new biologically active agentdelivery method and composition are disclosed. The compositions andmethods can be used to deliver agents such as therapeutics ordiagnostics which have been plagued with delivery problems, such asoligonucleotides, as well as traditional agents and can drasticallyreduce the effective dosages of each, increasing the therapeutic indexand improving bioavailability. This in turn can reduce drug cytotoxicityand side effects.

[0017] The invention employs conjugation of the biologic agent with afilmogenic protein which is formed as a protein shell microbubbleencapsulating an insoluble gas. The composition is prepared as anaqueous suspension of a plurality of the microbubbles for parenteraladministration. Conjugation of the biologic with albumin or other suchprotein encapsulated microbubbles can allow for targeted delivery of thebiologic to alternate including those which traditionally interact withthe protein.

[0018] Improved gas-filled microbubbles with enhanced stability and thusbetter delivery capabilities are achieved by forming the microbubbles inthe presence of a nitrogen-free environment. The nitrogen-freeenvironment makes microbubbles which are significantly smaller thanmicrobubbles obtained in a room air environment. These smallermicrobubbles are more stable than microbubbles manufactured in a roomair environment and result in improved delivery of the biologic.

DESCRIPTION OF THE FIGURES

[0019]FIG. 1 is a Lineweaver-Burke plot of the binding data for PESDAmicrobubbles with PS-ODN. The equilibrium dissociation constant K_(m)(calculated for the 7 concentrations which were run in duplicate) forthe binding to the microbubbles was 1.76×10⁻⁵M. (r²=0.999; Y-int=0.0566;7 concentrations). This is nearly within the range observed for bindinga 15mer PS-ODN with sequence 5′d(AACGTTGAGGGGCAT)-3′ (SEQ ID NO:1) tohuman serum albumin in solution of 3.7-4.8×10−5M previously reportedSrinivasan SK et al, “Characterization of binding sites, extent ofbinding, and drug interactions of oligonucleotides with albumin. AntisenRes. Dev. 5:131, 1995.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Ultrasonic imaging has long been used as a diagnostic tool to aidin therapeutic procedures. It is based on the principle that waves ofsound energy can be focused upon an area of interest and reflected toproduce an image. Generally an ultrasonic transducer is placed on a bodysurface overlying the area to be imaged and ultrasonic energy, producedby generating and receiving sound waves, is transmitted. The ultrasonicenergy is reflected back to the transducer where it is translated intoan ultrasonic image. The amount of characteristics of the reflectedenergy depend upon the acoustic properties of the tissues, and contrastagents which are echogenic are preferably used to create ultrasonicenergy in the area of interest and improve the imaging received. For adiscussion of contrast echographic instrumentation, see, DeJong and,“Acoustic Properties of Ultrasound Contrast Agents”, CIP-GEGEVENSKONINKLIJKE BIBLIOTHEEK, DENHAG (1993), pp. 120 et seq.

[0021] Contrast echocardiography has been used to delineate intracardiacstructures, assess valvular competence, and demonstrate intracardiacshunts. Myocardial contrast echocardiography (MCE) has been used tomeasure coronary blood flow reserve in humans. MCE has been found to bea safe and useful technique for evaluating relative changes inmyocardial perfusion and delineating areas at risk.

[0022] Ultrasonic vibration has also been used at therapeutic levels inthe medical field to increase the absorption of various medicaments. Forexample in Japanese Patent Kokai number 115591/1977 discloses thatpercutaneous absorption of a medicament is enhanced by ultrasonicvibration. U.S. Pat. Nos. 4,953,565 and 5,007,438 also disclose atechnique of percutaneous absorption of medicaments by the aid ofultrasonic vibration. U.S. Pat. No. 5,315,998 discloses a booster fordrug therapy comprising microbubbles in combination ultrasonic energy toallow the medicament to diffuse and penetrate. This discloses the use oftherapeutic levels of ultrasound for up to 20 minutes in contrast to theinvention which uses diagnostic levels of ultrasound with exposure formuch shorter time periods to achieve release of conjugated bioactiveagents.

[0023] Applicant has demonstrated that traditional diagnostic ultrasoundtherapy contrast agents can be used as a specific targeted deliverydevice to release therapeutic agents at the specifically designatedsites of interest thereby altering drug distribution. Surprisingly, thisobjective can be accomplished with the contrast agent alone and withoutthe use of any diagnostic ultrasound.

[0024] The pharmaceutical composition of the invention comprises aliquid suspension containing microbubbles of an insoluble gas having adiameter of 0.1 to 10 microns. The microbubbles are formed by entrappingmicrobubbles of a gas into a liquid. The microbubbles are made ofvarious insoluble gases such as fluorocarbon or sulfur hexafluoride gas.The liquid includes any liquid which can form microbubbles. Generallyany insoluble gas can be used. It must be gaseous at body temperatureand be nontoxic. The gas must also form stable microbubbles of averagesize of between about 0.1 and 10 microns in diameter when thepharmaceutical composition is sonicated to form microbubbles. Generallyperfluorocarbon gases such as perfluoromethane, perfluoroethane,perfluoropropane perfluorobutane, perfluoropentane are preferred. Ofthese gases, perfluoropropane and perfluorobutane are especiallypreferred because of their demonstrated safety for intraocular injectionin humans. They have been used in human studies for intraocularinjections to stabilize retinal detachments (Wong and Thompson,Opthamology 95:609-613). Treatment with intraocular perfluoropropane isconsidered to be the standard of care for treatment of this disorder.The gases must also have a diffusion coefficient and blood solubilitylower than nitrogen or oxygen which diffuse once in the internalatmosphere of the blood vessel.

[0025] Other inert gases such as sulfur hexafluoride are also useful inthe invention provided they have a diffusion coefficient and bloodsolubility lower than nitrogen or oxygen. The agent of the invention isformulated in a pharmaceutically effective dosage form for peripheraladministration to the host. Generally such host is a human host,although other mammalian hosts such as canine or equine can also besubject to this therapy.

[0026] The pharmaceutical liquid composition of the invention uses aliquid wherein the microbubbles are stabilized by a filmogenic proteincoating. Suitable proteins include naturally occurring proteins such asalbumin, human gamma globulin, human apotransferin, Beta lactose andurease. The invention preferably employs a naturally occurring proteinbut synthetic proteins may also be used. Preferred is human serumalbumin.

[0027] It is also optional to use an aqueous solution containing amixture of a pharmaceutically accepted saccharide e.g., dextrose, incombination with the earlier described protein. In a preferredembodiment the pharmaceutical liquid composition of the invention is thesonicated mixture of commercially available albumin (human), U.S.P.solution (generally supplied as 5% or 25% by weight sterile aqueoussolutions), and commercially available dextrose, U.S.P. for intravenousadministration. The mixture is sonicated under ambient conditions i.e.room air temperature and pressure and is perfused with an insoluble gas(99.9% by weight) during sonication.

[0028] In a most preferred embodiment the pharmaceutical liquidcomposition includes a two-fold to eight-fold dilution of 5% to 50% byweight of dextrose and a 2% to 10% by weight of human serum albumin.Exemplary of other saccharide solutions of the invention are aqueousmonosaccharide solution (e.g. having the formula C₆H₁₂O₆ such as thehexose sugars, dextrose or fructose or mixtures thereof), aqueousdisaccharide solution (e.g. having a formula C₁₂H₂₂O₁₁ such as sucrose,lactose or maltose or mixtures thereof), or aqueous polysaccharidesolution (e.g. soluble starches having the formula C₆H₁₀O₅(n) wherein nis a whole number integer between 20 and about 200 such as amylase ordextran or mixtures thereof.

[0029] The microbubbles are formed by sonication, typically with asonicating horn. Sonication by ultrasonic energy causes cavitationwithin the dextrose albumin solution at sites of particulate matter orgas in the fluid. These cavitation sites eventually resonate and producesmall microbubbles (about 7 microns in size) which are non-collapsingand stable. In general, sonication conditions which produceconcentrations of greater than about 4×10⁸m of between about 5 and about6 micron microbubbles are preferred. Generally the mixture will besonicated for about 80 seconds, while being perfused with an insolublegas, in the presence of room air.

[0030] A second method of preparation includes hand agitating 15±2 ml ofsonicated dextrose albumin with 8±2 ml of perfluorocarbon gas prior tosonication. Sonication then proceeds for 80±5 seconds.

[0031] In a preferred embodiment, the microbubbles are formed in a 100′oxygen or a nitrogen-free environment. Microbubbles formed in theseenvironments are significantly smaller than those formed in the presenceof room air. These smaller microbubbles are more stable and result inimproved delivery of biologics.

[0032] The inventors became aware of the advantages of using anitrogen-free environment or 100% oxygen through the observation thatgas-filled microbubbles produced better ultrasound contrast followingvenous injection than room air filled microbubbles. One reason for thisimproved contrast is the prolonged survival of the gas-filledmicrobubbles following intravenous injection. In comparison, room airfilled microbubbles of comparable size are rapidly destroyed followingvenous injection because of rapid diffusion of the soluble gases out ofthe microbubble. Computer simulations, however, have shown that thesesoluble gases still affect the size of gas-filled microbubbles in blood,thereby affecting their ultrasound characteristics. Burkard, M. E. etal. (1994), Oxygen transport to tissue by persistent bubbles: theory andsimulations. J Appl Physiol 2874-8. These models have theorized thatblood nitrogen plays an important role in preventing the outwarddiffusion of the gas within the microbubble.

[0033] It was postulated that by enhancing microbubble oxygen content(thus lowering partial pressures of nitrogen within the microbubble),they could prolong microbubble survival in blood. The presence of anitrogen-free environment was found to produce substantially smallermicrobubbles which are more stable in the bloodstream. This results inimproved contrast and drug delivery.

[0034] These microbubble sizes are particularly ideal since amicrobubble must have a mean diameter of less than 10 microns andgreater than 0.1 to be sufficient for transpulmonary passage, and mustbe stable enough to prevent significant diffusion of gases within themicrobubble following intravenous injection and during transit to thetarget site.

[0035] As used herein the term “nitrogen free” shall mean a nitrogencontent which is less than that of room air such that the partialpressure of nitrogen is gas-filled microbubbles formed by sonication islower than that achieved from sonication with room air (typically about70-80% nitrogen).

[0036] The microbubbles are next incubated with the medicament so thatthe medicament becomes conjugated with the microbubble. Quiteunexpectedly it was demonstrated that filmogenic proteins in the form ofmicrobubbles as previously used in contrast agents retain their abilityto bind medicaments. This is surprising because traditionally it wasthought that in the formation of microbubble contrast agents the proteinsphere was made of denatured protein. Applicant has demonstrated thatwhen an insoluble gas instead of air is used for the microbubble, muchof the sonication energy is absorbed by the gas and the protein retainsits binding activity. Air filled microbubbles do not retain theirbinding capabilities and cannot be used in the method of the invention.

[0037] The therapy involves the use of a pharmaceutical compositionconjugated to a protein microbubble of a diameter of about 0.1 to 10microns. The invention uses agents traditionally used in diagnosticultrasound imaging.

[0038] Therapeutic agents useful in the present invention are selectedvia their ability to bind with the filmogenic protein. For example ifthe filmogenic protein is albumin, the therapeutic or diagnostic agentcan include oligonucleotides (such as antisense or antigen oligos),polynucleotides (such as retroviral, adenoviral, plasmid vectors orprobes), or ribozymes all of which can bind with albumin and as such canform a conjugation with the microbubble. A list of drugs which bind toalbumin at site 1 (which retains its binding capacity) and thus would beuseful in the methods and compositions of the present invention in thealbumin embodiment follows: Drug % Albumin Binding Drug Class Naproxen99.7 NSAID⊕ Piroxicam 99.3 NSAID⊕ Warfarin 99.0 Anticoagulant Furosemide98.8 Loop diuretic Phenylbutazone 96.1 NSAID⊕ Valproic Acid 93.0Antiepileptic Sulfisoxazole 91.4 Sulfonimide Antibiotic Ceftriaxone90-95* Third Generation cephalosporin antibiotic Miconazole 90.7-93.1*Antifungal Phenytoin 89.0 Antiepileptic

[0039] Other drugs which bind with albumin particularly at site 1 wouldalso be useful in this embodiment and can be ascertained by those ofskill in the art through Drug Interaction and Pharmacology testsstandard to those of skill in the art such as “Drug Information or“Facts and Comparisons” published by Berney Olin updated every quarter.Other such references are widely available in the are. Assays fordetermination of appropriate protein-therapeutic combinations aredisclosed herein and can be used to test any combination for its abilityto work with the method of the invention.

[0040] According to a preferred embodiment of the invention, proteincoated microbubbles of insoluble gas have been found to form stableconjugates with oligonucleotides. The oligo conjugated bubbles are thenintroduced to the animal and the protein coating directs the conjugatedagent to sites of interaction. Ultimately as the bubble dissipates theagent will be released at the tissue site.

[0041] This is of particular relevance to oligonucleotide andpolynucleotide therapy as the primary hurdle to effective anti-sense,anti-gene, or even gene therapy employing viral or plasmid nucleotidedelivery is the ability of the therapeutic to reach the target site athigh enough concentrations to achieve a therapeutic effect. Therapeuticsites can include such things as the location of a specific tumor, anorgan which due to differential gene activation expresses a particulargene product, the site of an injury or thrombosis, a site for furtherprocessing and distribution of the therapeutic etc. Generally the targetsite is selected based upon the bioprocessing of the filmogenic protein.For example the kidneys and liver take up albumin and albuminmicrobubbles can be used to specifically direct the administration ofconjugated bioactive agents to these areas. The metabolism andbioprocessing of other filmogenic proteins can be easily obtainedthrough standard pharmacologic texts such as

Basic and Clinical Pharmacology

by Bertram G. Katzung the relevant disclosure of which is incorporatedby reference.

[0042] The method preferred for practicing the delivery therapy of theinvention involves obtaining a pharmaceutical liquid agent of theinvention, introducing said agent into a host by intravenous injection,intravenously (i.v. infusion), percutaneously or intramuscularly. Themicrobubble is then processed in the animal and is taken up andinteracted with according to the filmogenic protein which coats themicrobubble. Ultimately the bubble dissipates delivering the bioactiveat the site of processing of the protein.

[0043] It has been previously shown by applicants that microbubbleconjugation of bioactive agents can be used in targeted deliveryprotocols with delivery of the biologic upon application of ultrasoundto the target site, causing cavitation of the microbubble and ultimaterelease of the biologic at the site in interaction with the ultrasoundfield. Quite unexpectedly, applicant has now discovered that applicationof ultrasound is not necessary for the targeted delivery of biologics tosites of bioprocessing of the protein coating. The protein traffics themicrobubble and conjugate to sites of processing and as the bubblesdissipate the oligo or other biologic is released to interact with thesite allowing for a fraction of the biologic to achieve the samebiological effect.

[0044] In a preferred embodiment the agent of the invention is aperfluorocarbon enhanced sonicated dextrose albumin solution comprisedof a sonicated three-fold dilution of 5% human serum albumin with 5%dextrose. During sonication, the solution is perfused withperfluorocarbon gas for about 80 seconds which lowers the solubility anddiffusivity of the microbubble gas. The resulting microbubbles areconcentrated at room temperature for at least about 120±5 minuteswherein the excess solution settles in the sonicating syringe. Themicrobubbles are then exposed to a therapeutic agent and allowed tointeract such that the agent becomes conjugated to the microbubbles.Next the conjugated microbubbles are transferred to a sterile syringeand injected parenterally into a mammal, preferably near the target siteof activity of the agent.

[0045] Methods of ultrasonic imaging in which microbubbles formed bysonicating an aqueous protein solution are injected into a mammal toalter the acoustic properties of a predetermined area which is thenultrasonically scanned to obtain an image for use in medical proceduresis well known. For example see U.S. Pat. No. 4,572,203, U.S. Pat. No.4,718,433 and U.S. Pat. No. 4,774,958, the contents of each of which areincorporated herein by reference.

[0046] It is the use of these types of contrast agents as apharmaceutical composition as part of a targeted delivery system that isthe novel improvement of this invention. The use of a nitrogen-freeenvironment in the manufacture of the contrast agents is also a novelimprovement in the effectiveness of the contrast agent in myocardialimaging.

[0047] The invention has been shown to drastically improve theefficiency and therapeutic activity by altering biodistribution ofseveral drugs including, most notably, antisense oligonucleotides whichhave been traditionally plagued with ineffective pharmacologicparameters, including high clearance rate and toxicity.

[0048] This is particularly significant as the microbubble-therapeuticagent therapy can reduce any toxic effects of persons who perhaps couldnot tolerate certain therapeutics at doses and concentrations necessaryto achieve a beneficial result.

[0049] The protein substance such as human serum albumin is easilymetabolized within the body and excreted outside and hence is notharmful to the human body. Further gas trapped within the microbubblesis extremely small and is easily dissolved in blood fluid,perfluoropropane and perfluorobutane have long been known to be safe inhumans. Both have been used in humans for intra ocular infections tostabilize retinal detachments. Wong and Thompson, Ophthalmology95:609-613. Thus the anti thrombosis agents of the invention areextremely safe and nontoxic for patients.

[0050] The invention is particularly useful for delivery of nucleotidesequences in the form of gene therapy vectors, or anti-sense ofanti-gene type strategies to ultimately alter gene expressions in targetcells.

[0051] Antisense oligonucleotides represent potential tools in researchand therapy by virtue of their ability to specifically inhibit synthesisof target proteins. A major theoretical advantage of these oligos istheir potential specificity for binding to one site in the cell.According to one embodiment of the invention a synthetic oligonucleotideof at least 6 nucleotides, preferably complementary to DNA (antigene) orRNA (antisense), which interferes with the process of transcription ortranslation of endogenous proteins is presented.

[0052] Any of the known methods for oligonucleotide synthesis can beused to prepare the oligonucleotides. They are most convenientlyprepared using any of the commercially available, automated nucleic acidsynthesizers, such as applied biosystems, Inc., DNA synthesizer (Model380B). According to manufacturers protocols using phosphoroamiditechemistry. After biosystems (Foster City, Calif.). Phosphorothioateoligonucleotides were synthesized and purified according to the methodsdescribed in Stek and Zahn J. Chromatography, 326:263-280 and in AppliedBiosystems, DNA Synthesizer, User Bulletin, Models380A-380B-381A-391-EP, December 1989. The oligo is introduced to cellsby methods which are known to those of skill in the art. See Iverson, etal., “Anti-Cancer Drug Design”, 1991, 6531-6538, incorporated herein byreference.

[0053] Traditional limitations of oligonucleotide therapy have beenpreparation of the oligonucleotide analogue which is substantiallyresistant to the endo and exonucleases found in the blood and cells ofthe body. While unmodified oligos have been shown to be effective,several modifications to these oligos has helped alleviate this problem.

[0054] Modified or related nucleotides of the present invention caninclude one or more modifications of the nucleic acid bases, sugarmoieties, internucleoside phosphate linkages, or combinations ofmodifications at these sites. The internucleoside phosphate linkages canbe phosphorothioate, phosphoramidate; methylphosphonate,phosphorodithioate and combinations of such similar linkages (to producemix backbone modified oligonucleotides). Modifications may be internalor at the end(s) of the oligonucleotide molecule and can includeadditions to the molecule of the internucleoside phosphate linkages,such as cholesterol, diamine compounds with varying numbers of carbonresidues between the amino groups, and terminal ribose, deoxyriboase andphosphate modifications which cleave, or crosslink to the oppositechains or to associated enzymes or other proteins which bind to thegenome.

[0055] These modifications traditionally help shield the oligo fromenzymatic degradation within the cell. Any of the above modificationscan be used with the method of the invention. However, in preferredembodiment the modification is a phosphorothioate oligonucleotide.

[0056] The following examples are for illustration purposes only and arenot intended to limit this invention in any way. It will be appreciatedby those of skill in the art, that numerous other protein-bioactiveagent combinations can be used in the invention and are evencontemplated herein. For example, if the filmogenic protein istransferrin, the bioactive agent could be any transferrin bindingpharmacologic.

[0057] In all the following examples, all parts and percentages are byweight unless otherwise mentioned, all dilutions are by volume.

EXAMPLE 1 Phosphorothioate Oligonucleotide Synthesis

[0058] Chain extension syntheses were performed on a 1 μmole columnsupport on an ABI Model 391 DNA synthesizer (Perkin Elmer Foster City,Calif.) or provided by Lynx Therapeutics, Inc. (Hayward Calif.). The 1micromole synthesis employed cyanoethyl phosphoroamidites andsulfurization with tetraethylthiuram disulfide as per ABI user Bulletin58.

[0059] Radiolabeled oligonucleotides were synthesized as hydrogenphosphonate material by Glen Research (Bethesda, Md.) The uniformly³⁵S-labeled PS-ODN with sequences 5′-TAT GCT GTG CCG GGG TCT TCG GGC 3′(24-mer complementary to c-myb) (SEQ ID NO:2) and 5′ TTAGGG 3′ (SEQ IDNO:3) were incubated in a final volume of 0.5 ml with theperfluorocarbon-exposed sonicated dextrose albumin microbubble solutionfor 30 minutes at 37° C. The solutions were allowed to stand so that thebubbles could rise to the top and 100 microliters were removed from theclear solution at the bottom and 100 microliters were removed from thetop containing the microbubbles.

[0060] Preparation of Microbubble Agent

[0061] Five percent human serum albumin and five percent dextrose wereobtained from a commercial source. Three parts of 5% dexticse and onepart 5% human serum albumin (total 16 milliliters) were drawn into a35-milliliter Monojet syringe. Each dextrose albumin sample was handagitated with 8±2 milliliters of either a fluorocarbon gas(decafluorobutane; molecular weight 238 grams/mole) or 8±2 millilitersof room air, and the sample was then exposed to electromechanicalsonication at 20 kilohertz for 80±5 seconds. The mean size of fourconsecutive samples of the perfluorocarbon-exposed sonicated dextrosealbumin (PESDA) microbubbles produced in this manner, as measured withhemocytometry was 4.6±0.4 microns, and mean concentration, as measuredby a Coulter counter was 1.4×10⁹ bubbles/milliliter. The solution ofmicrobubbles was then washed in a 1000 times volume excess of 5%dextrose to remove albumin which was not associated with themicrobubbles. The microbubbles were allowed four hours to rise. Thelower solution was then removed leaving the washed foam. The washed foamwas then mixed with 0.9% sodium chloride.

[0062] Binding Assays

[0063] The radioactive 24-mer PS-ODN was added to a washed solution ofPESDA and room air sonicated dextrose albumin (RA-SDA) microbubbles at aconcentration of 5 nM. Non-radioactive PS-ODN 20-mer was added to tubescontaining radioactive 24-mer in a series of increasing concentrations(0, 3.3, 10, 32.7, 94.5, 167, and 626 μM). The suspension of bubbles ismixed by inversion and incubated at 37° C. for 60 minutes.

[0064] Measurement of Radioactivity

[0065] Radioactivity in solutions were determined by liquidscintillation counting in a liquid scintillation counter (model LSC7500;Beckman Instruments GmbH, Munich, Germany). The sample volume was 100 μlto which 5 ml of Hydrocount biodegradable scintillation cocktail wasadded and mixed. Samples were counted immediately after each experimentand then again 24 hours later in order to reduce the influence ofchemiluminescence and of quenching.

[0066] Flow Cytometry

[0067] The uniformity of room air versus perfluorocarbon-containingsonicated dextrose albumin microbubble binding of PS-ODN was determinedby flow cytometry. A solution of microbubbles was washed in a 1000 foldexcess volume of sterile saline. Three groups of samples were preparedin triplicate as follows; Group A (control) in which 100 μl ofmicrobubbles were added to a 900 μL of saline, Group B in which 100 μlof microbubbles were added to 900 μL of saline and 2 μL of FITC-labeled20-mer was added (final 20-mer concentration is 151 nM), and group C inwhich 100 μL of microbubbles were added to 800 μL of saline, 2 μL ofFITC-labeled 20-mer and 100 μL of unlabeled 20-mer(final concentrationis 151 nM). The incubations were all conducted for 20 minutes at roomtemperature.

[0068] Washed microbubble suspensions were diluted in sterile saline(Baxter) and then incubated with FITC-labeled PS-ODN. Flow cytometricanalysis was performed using a FACStar Plus (Becton Dickinson) equippedwith t 100 mW air-cooled argon laser and the Lysis II acquisition andanalysis software. List mode data were employed for a minimum of 10⁴collected microbubbles and independent analysis a for each sample.

[0069] Study Protocol

[0070] A variable flow microsphere scanning chamber was developed forthe study which is similar to that we have described previously Mor-AviV., et al “Stability of Albunex microspheres under ultrasonicirradiation; and in vitro study. J Am Soc Echocardiology 7:S29, 1994.This system consists of a circular scanning chamber connected to aMasterflex flow system (Microgon, Inc., Laguna Hills California) Thescanning chamber was enclosed on each side by water-filled chambers andbound on each side by acoustically transparent material. ThePS-ODN-labeled PESDA microbubbles (0.1 milliliters) were injected as abolus over one second proximal to the scanning chamber which then flowedthrough plastic tubing into a tap water-filled scanning chamber at acontrolled flow rate of 100 ml/min. As the bubbles passed through thescanning chamber, the scanner (2.0 Megahertz) frequency, 1.2 Megapascalspeak negative pressure) was set to either deliver ultrasound at aconventional 30 Hertz frame rate or was shut off. Following passagethrough the scanning chamber, the solution was then passed through thesame size plastic tubing into a graduated cylinder. The first 10milliliters was discarded. Following this, the next 10 milliliters wasallowed to enter into a collection tube. The collection tube containingthe effluent microbubbles was allowed to stand in order to separatemicrobubbles on the top from whatever free oligonucleotide existed inthe lower portion of the sample. Drops from both the upper and loweroperation of the effluent were then placed upon a hemocytometer slideand analyzed using a 10× magnification. Photographs of these slides werethen made and the number of microbubbles over a 36 square centimeterfield were then hand-counted. The upper and lower layers of theremaining effluent were then used for analysis of oligonucleotidecontent using flow cytometry in the same manner described below.

[0071] Microbubble samples exposed to the various oligonucleotidesolution were mixed 15 (v/v) with a solution of formamide and EDTA andheated to 95° C. for 5 minutes. These samples were then examined on anApplied Biosystems Model 373A DNA sequencer with e 20% polyacrylamidegel. The data were acquired with GeneScanner software so thatfluorescence intensity area under the curve could be determined.

EXAMPLE 2 Phosphorothioate Oligonucleotide Binding of PESDA VersusRA-SDA Microbubbles

[0072] The partitioning of PS-ODN to PESDA microbubbles top layer) andnon-bubble washed (albumin-free) and unwashed (non-bubble albumincontaining) lower layers as counted by liquid scintillation counting aredemonstrated in Table 1. TABLE 1 OLIGONUCLEOTIDES BINDING TO ALBUMIN OFPESDA MICROBUBBLES TOP BOTTOM RATIO N cpm/μl cpm/μl T/B BUBBLES IN THEPRESENCE OF FREE ALBUMIN TTAGGG 6  125 ± 6.4 92.3 ± 6.4 1.35 c-myb 6 94.1 ± 17.6 77.3 ± 1.2 1.35 WASHED BUBBLES (NO FREE ALBUMIN) TTAGGG 6 210 ± 10.8  126 ± 8.7 1.67 c-myb 6 200.3 ± 37.4  92.7 ± 15.7 2.16

[0073] These data indicate that albumin in the unwashed solution whichis not associated with the microbubble will bind to the PS-ODN so thatthe partitioning of PS-ODN is equivalent between microbubbles (toplayer) and the surrounding solution (lower layer; p=HS). Removal ofnon-microbubble associated albumin (Washed Bubbles in Table 1) does notshow a significant partitioning of the PS-ODNs with the PESDAmicrobubbles (1.67 for TTAGGG PS-ODN and 2.16 for c-myb PS-ODN). Therecovery of total radioactivity in the experiments reported in Table 1is 96′ of the radioactivity added which is not significantly differentfrom 100%.

[0074] The affinity of binding of PS-ODN to washed microbubbles wasevaluated by addition of increasing amounts of excess non-radioactivePS=ODN as a competing ligand for binding sites. In this case a 20merPS-ODN with sequence 5′-d(CCC TGC TCC CCC CTG GCT CC)-3′ (SEQ ID NO:4)was employed to displace the radioactive 24mer. Albumin proteinconcentrations in the washed microbubble experiments was 0.28±0.04 mg/mlas determined by the Bradford Assay Bradford M et al “A Rapid andSensitive Method for the quantification of microgram quantities ofprotein utilizing the principle of protein-dye binding” anal. Bioche,.72:248, 1976. The observed binding data are presented as a LineweaverBurke plot in FIG. 1. The equilibrium dissociation constant K_(m)(calculated for the 7 concentrations which were run in duplicate) forthe binding to the microbubbles was 1.76×10⁻⁵ M.

[0075] The distribution of FITC-labeled microbubbles is provided inTable 2. TABLE 2 DISTRIBUTION OF OLIGONUCLEOTIDE (PS-ODN) BOUNDMICROBUBBLES Control 151 nM Excess PS-ODN FITC PS-ODN Unlabeled ODN No.PE MI PE MI PE MI 1 99.5 2.38 98.9 2109.8 97.8 1753.1 2 99.3 4.07 99.12142.3 98.7 1710.9 3 99.4 3.52 99.1 2258.5 99.3 1832.2 mean 3.23 21701765 ±SE ±0.50 ±46¹ ±36^(1,2)

[0076] The significant decrease in mean fluorescence intensity in thesamples containing excess unlabeled PS-ODN indicates the binding tomicrobubbles is saturable. Consequently, since the binding is saturable,the nonspecific interactions of PS-ODN with the microbubble surface arelimited. A Gaussian distribution of PS-ODN to washed PESDA microbubblesindicated that the albumin on these microbubbles had retained itsbinding site for the oligonucleotide. The absence of a Gaussiandistribution for washed RA-SDA indicated loss of albumin binding site 1for this oligonucleotide occurred during sonication of thesemicrobubbles. For a discussion of albumin binding characteristicsparticularly as they relate to oligonucleotides see Kumar, Shashi et al“Characterization of Binding Sites, Extent of Binding, and DrugInteractions of Oligonucleotides with Albumin” Antisense Research andDevelopment 5: 131-139 (1995) the disclosure of which is herebyincorporated by reference.

[0077] From the foregoing it can be seen that, PS-ODN binds to thealbumin in PESDA microbubbles, indicating that the binding site 1 onalbumin is biologically active following production of these bubbles byelectromechanical sonication. This binding site affinity is lost whenthe electromechanical sonication is performed only with room sir.Further, removal of albumin not associated with PESDA microbubbles bywashing shows a significant partitioning of the PS-ODNs with themicrobubbles (Table 1). These observations demonstrate that albumindenaturation does not occur with perfluorocarbon-containing dextrosealbumin solutions during sonication as has been suggested withsonication in the presence of air. The retained bioactivity of albumin(especially at site 1) in PESDA microbubbles was confirmed by theaffinity of binding of PS-ODN to washed PESDA microbubbles in thepresence of increasing amounts of excess non-radioactive PS-ODN as acompeting ligand for binding sites (Table 2). The significant decreasein mean fluorescence intensity in the samples containing excessunlabeled PS=−ODN indicates the binding to microbubbles is saturable.

EXAMPLE 3 Altered Biodistribution Via Microbubble Delivery of AntisenseOligos

[0078] According to the invention antisense phosphorothioateoligonucleotides were designed to the cytochrome P450 IIB1 gene sequenceto alter the metabolism of Phenobarbital. The oligonucleotides wereconjugated to perfluoropropane exposed sonicated dextrose albuminmicrobubbles (PESDA) as earlier described and delivered to ratsintravenously. The oligonucleotide was synthesized according to the ratcytochrome P450 IIB1 known sequence and had the following sequence:GGAGCAAGATACTGGGCTCCAT (SEQ ID NO:5) AAAGAAGAGAGAGAGCAGGGAG (SEQ IDNO:6)

[0079] Male Sprague-Dawley rats (Sasco, Omaha), were used and weighedbetween 210 to 290 grams for all studies. They were housed in animalquarters at the University of Nebraska Medical Center, AAALAC approvedanimal resource facility. The animals were exposed to 12 hour light/darkcycle and allowed access to Purina rat chow and tap water ad libitum.

[0080] Rats in groups with PB were injected intraperitoneally withphenobarbital (Mallinckrodt, St. Louis) at 80 ml/kg/day×2 days. The PBinjections were given simultaneously with the ODN-microbubbleinjections. Phosphorothioate ODN injections were 1 ml/kg/day×2 days.Sleep times were measured 48 hours after the first injection. The ratswere injected intraperitoneally with 100 ml/kg hexobarbital (Sigma, St.Louis), paired fresh daily. The volume of this injection is 1 ml/kg bodyweight.

[0081] Each rat was injected with 100 mg/kg of hexobarbitalintraperitoneally. The animals were placed on their backs to insure thatthey were still under sedation from the hexobarbital. Sleep time isdefined as the time they are placed on their backs to the time when theyroll over. The sleep times listed are the mean of each animal in thegroup±standard deviation.

[0082] Results indicate that delivery of the oligonucleotide conjugatedmicrobubbles greatly improved efficacy of the drug. Rats given {fraction(1/20)}th dose of oligo experienced a sleep time of more than 50minutes. This is compared to non microbubble conjugated oligo with anapproximate sleep time of 13 minutes.

[0083] Rats were ultimately sacrificed using ethyl ether and microsomeswere prepared as described by Franklin and Estabrook (1971). Livers wereperfused with 12 ml of 4% saline via the portal vein and then removedfrom the animal. The livers were minced, homogenized in 0.25 M sucrose(Sigma) and centrifuged at 8000×g for 20 minutes at 4° C. in a SorvallRC2-B centrifuge (Dupont, Wilmington, Del.). The supernatant was savedand resuspended in a 0.25 M sucrose and centrifuged at 100,000×g for 45minutes at 4° C. in a Sorvall OTD55B ultracentrifuge (Dupont). Thepellet was resuspended in 1.15% KCL (Sigma) and centrifuged at 100,000×gfor 1 hour at 4° C. with the final pellet resuspended in an equal volumebuffer (10 mM Tris-acetate, 1 mM EDTA, 20% glycerol; Sigma) and frozenat −80° C.

[0084] Protein concentrations were determined by Bradford assay(Bradford, 1976). 80 μl aliquots of homogenate were added to a 96 wellplate (Becton, Dickinson Labware, Lincoln Park, NJ). 20 μl of Bradfordreagent (Bio-Rad Richmond, Calif.) was then added and the plates read at595 nm on the microplate reader (Molecular Devices, Newport Minn.). Thedata was compared to standard curve generated with known concentrationsof bovine serum albumin (Sigma).

[0085] CYP IIB1 content was determined by pentoxyresorufinO-dealkylation (PROD) activity (Burke et al. 1985). For each microsomalsample, 1 mg protein in 1 ml 0.1 M potassium phosphate buffer, 1 ml 2 μM5-pentoxyresorufin (Pierce, Rockford, Ill.), and 17 μl 60 mM NADPH weremixed and incubated for 10 minutes at 37° C. The mixture was then addedto a 2 ml cuvette and read on a RF5000U spectrofluorophotometer(Shimadzu, Columbia, Md.) using an excitation wavelength of 530 nm andemission wavelength of 585 nm. Concentrations of unknowns werecalculated from a standard curve of resorufin (Pierce, Rockford, Ill.)standards. Results were recorded in nmol resorufin/mg protein/min.

[0086] Direct measurement of CYP IIB1 protein was determined by an ELISAassay using an antibody directed the CYP IIB1 protein (Schuurs and VanWeeman, 1977). 50 μg of liver per well were plated in 100 μl 0.35%sodium bicarbonate buffer overnight on a 96 well nunc-immuno plate(InterMed, Skokie, IL). The microsomes were washed 3× with 1% bovineserum albumin in PBS (PBS/BSA) and incubated for 1 hr at 37° C. with 200μl PBS/BSA. The PBS/BSA was removed and 50 μl of CYP IIB1 antibody(Oxygene, Dallas) was added and incubated for 1 hour at 37° C. Themicrosomes were washed 5× with saline/tween 20 (Sigma) and had 50 μlhorseradish peroxidase antibody (Bio-rad) added. The microsomes wereincubated for 1 hour at 37° C., washed 5× with saline/tween 20 and twicewith 85% saline. 100 μl of horseradish peroxidase substrate (Kirkegaard& Perry Labs, Gaithersburg, Md.) was added and the plate readcontinuously in a microplate reader (Molecular Devices) at 405 nm for 1hour. Results were recorded as horseradish peroxidase activity inmOD/min.

[0087] Results demonstrated that the oligo conjugated microbubblesdirected the oligo to the liver and kidney. These are site ofphenobarbitol metabolism. As described earlier, 100 mg/kg HB wasinjected i.p. to each animal at the end of 2 days of treatment with PBand/or the ODNs. Control rats had a sleep time of about 23 minutes. PBhad a significant reduction in sleep time to about 11.4±4.5 minutes. PBstimulates CYP IIB1 mRNA, as a result, hexobarbital which ishydroxylated by CYP IIB1 is more quickly metabolized and its sedativeeffect reduced.

EXAMPLE 4 Comparison Study of Microbubbles Formed in Nitrogen-FreeVersus Room Air Environments

[0088] The perfluorocarbon containing microbubbles (PCMB) used for thisstudy were perfluorocarbon exposed sonicated dextrose albumin. Toprepare these microbubbles, one part 5% human serum albumin and threeparts 5% dextrose (total of 16 ml) were combined in a 35 ml Monojectsyringe (Sherwood Medical, St. Louis, Mo.). This sample was thenhand-agitated with 8 ml of fluorocarbon gas (decafluorobutane; MW 238g/mol). Following the agitation, the sample underwent electromechanicalsonication for 80±2 seconds.

[0089] For in vivo studies, the 80 second sonication process wasperformed in two different environments: either room air or 100% oxygen(nitrogen-free environment) was blown into interface between thesonicating horn and perfluorocarbon dextrose albumin solution duringsonication. Each of the samples prepared in this manner had microbubblesize determined with hemocytometry and concentration determined with aCoulter counter (Coulter Electronics, Inc. Hiahleah, Fla.).

[0090] In Vitro Scanning Chamber Set Up

[0091] The scanning chamber system consisted of a 35 ml cylindricalscanning chamber connected to a peristaltic Masterflex flow system(Microgon, Inc., Laguna Hills, Calif.). Enclosed on both sides of thescanning chamber are cylindrical saline filled chambers, bound byacoustically transparent latex material that is 6.6 microns in thickness(Safeskin, Inc.; Boca Raton, Fla.). Pressure within the scanning chamberduring ultrasound exposure was measured with a pressure transducerplaced just proximal to the scanning chamber (model 78304A; HewlettPackard Co., Andover, Mass.), and averaged 7±3 mm Hg throughout all ofthe trials.

[0092] Two different 2.0 Megahertz ultrasound transducers were used forthe in vitro studies (Hewlett Packard 1500; Andover, Mass.; and HDI3000, Advanced Technology Laboratories, Bothell, Washington). The peaknegative pressure generated by the Hewlett Packard transducer was 1.1megapascals, while it was 0.9 megapascals for the HDI 3000 scanner.Imaging depth for all studies was 8.2 centimeters, and the focal pointfor both transducers was 8 centimeters. The frame rate for eachtransducer was either conventional (30-42 Hertz) or intermittent (1Hertz). All images from the scanning chamber were recorded on highfidelity videotape.

[0093] In Vitro Protocol

[0094] Arterial blood during room air inhalation was taken from fourdogs and three healthy pigs just prior to sacrifice. In four of theanimals, additional arterial blood was obtained after the animal hadinhaled 100% oxygen for a minimum of 10 minutes. The blood was collectedin 60 ml heparinized syringes, and kept in a warm water bath at 37° C.until injected into the scanning chamber. Immediately before injectionof the blood into the scanning chamber, 0.2 ml of PCMB were injected viaa stopcock into the 60 ml syringe of blood, and mixed gently byinverting and rolling the syringe by hand.

[0095] Once the PCMB were well-mixed with the blood, the tip cap wasremoved from the syringe, and the syringe was connected to plastictubing (3.5 mm in diameter) proximal to the Masterflex flow system. At aflow rate of 50 ml/minute, the contrast filled blood flowed from thesyringe into the tubing and then into the scanning chamber. Once thechamber was filled, the closed stopcock connecting the scanning chamberto the plastic tubing distal to the chamber was opened, and ultrasoundexposure (intermittent at 1 Hertz frame rate or conventional at 30-45Hertz) was initiated. The effluent blood after ultrasound exposureflowed out of the scanning chamber into tubing which was connected to agraduated cylinder. The first 10 ml of blood was discarded, and the next15 ml of blood that flowed from the chamber was collected in three 5 mlaliquots into inverted capped syringes. Three minutes following thecollection of the last 5 ml sample, a tuberculin syringe was dipped intothe top level of the effluent blood and a drop placed on a hemocytometerslide; this length of time was chose to allow the microbubbles in theeffluent blood to rise to the top and be collected. The hemocytometerslide was then examined at 40× magnification with a light microscope(Olympus BH-2, Olympus America Inc., Woodbury, N.Y.) and the fieldcontaining the highest concentration of microbubbles was photographed onthe hemocytometer field.

[0096] The photos were later enlarged on a photocopy machine, and a 25cm² field was chosen to analyze concentration and the diameter of eachmicrobubble in the field. The mean diameter was calculated, thus givingmean microbubble size. Concentration was determined by counting thetotal number of bubbles in the entire slide. Microbubble concentrationmeasured with this technique has correlated very closely with Coultercounter measurements, and size measurements with this technique havebeen calibrated with a known 5 micron sphere (Coulter Size StandardsNominal 5 Am Microspheres, Miami, Fla.).

[0097] In Vivo Studies

[0098] The inventors subsequently tested the effect of a nitrogen-freeenvironment within the perfluorocarbon microbubble by randomly givingintravenous injections of PCMB of the same concentration of microbubblesexposed to either 100% oxygen (O2 PCMB) or room air (RA PCMB) duringsonication. Imaging was performed with a 1.7 megahertz harmonictransducer (HDI 3000; Advanced Technology Laboratories; Bothell,Washington). Transducer output was set to 0.3-0.8 megapascals, and keptconstant for all comparisons of videointensity from the two differentmicrobubble samples. Frame rates for comparison of background subtractedmyocardial videointensity were either 43 Hertz (conventional) or 10Hertz (intermittent). All procedures were approved by the InstitutionalAnimal Care and Use Committee and was in compliance with the Position ofthe American Heart Association on Research Animal Use.

[0099] The bolus injections of RA PCMB and O₂ PCMB were either 0.0025 or0.005 ml/kg, since the concentrations of each microbubble were the same.Peak anterior and posterior myocardial videointensity were measured fromdigitized super VHS videotape images (Maxell, Japan) obtained off-lineusing a Tom-Tech review station (Louisville, Colo.). This quantifiesvideointensity over a 1-255 gray scale range. The region of interest wasplaced in the mid myocardium of each segment.

[0100] In addition to this quantitative analysis, the visual assessmentof regional myocardial contrast enhancement in the anterior, septal,lateral, and posterior regions from the short axis view was made by twoindependent reviewers. Each region was assigned a 0 (no myocardialcontrast), 1+ (mild myocardial contrast enhancement) or 2+ (brightmyocardial contrast enhancement which approached cavity intensity).

[0101] Statistical Analysis

[0102] An unpaired t test was used to compare microbubble size andconcentration of the PCMB samples exposed to different gases duringsonications. This was also used to compare differences in peakmyocardial videointensity in the in vivo studies. If the data was notnormally distributed, a non-parametric test was performed. Comparisonsof visual myocardial contrast enhancement following intravenous O2 PCMBand RA PCMB were made with continency tables (Fisher's Exact Test). A pvalue less than 0.05 was considered significant.

[0103] A coefficient of variation was used to measure interobservervariability in the measurements of microbubble size and concentration inthe in vitro studies. A mean difference between independent reviewerswas used to compare interobserver variations in peak myocardialvideointensity.

[0104] Results

[0105] Table 3 demonstrates differences in mean microbubble size forPCMB after exposure to ultrasound in arterial blood (room air and 100%oxygen). When PCMB were exposed to 100% oxygenated arterial blood, therewas a significant decrease in mean microbubble size after insonation(p=0.01). The smaller microbubble size was seen both after intermittentimaging (7.3±3.7 microns room air vs. 6.4±3.2 microns 100% oxygen) andafter conventional imaging (7.5±3.5 microns room air vs. 6.3±3.0 microns100% oxygen).

[0106] Microbubble concentration decreased significantly after exposureto conventional frame rates when compared to intermittent imaging inroom air arterial blood (Table 3). However, conventional frame rates atthe same transducer output did not destroy as many PCMB when they werein oxygenated arterial blood. TABLE 3 COMPARISON OF EFFLUENT PESDAMICROBUBBLE SIZE AFTER EXPOSURE TO DIFFERENT ULTRASOUND FRAME RATES INROOM AIR AND 100% OXYGENATED ARTERIAL BLOOD MB size MB Conc. (No./hpf)(μm) Conv Inter Arterial 7.4 ± 3.6 6 ± 8 16 ± 11

Arterial +O₂ 6.3 ± 3.1 11 ± 9  14 ± 9* 

[0107] In Vivo Studies

[0108] A total of six comparisons of peak myocardial videointensitybetween O₂ PCMB and RA PCMB were made in the three dogs. In Table 4, itcan be seen that prior to injection, the PCMB sonicated in the presenceof 100% oxygen were similar in size and concentration to PCMB sonicatedin the presence of room air. However, in all three dogs, the peakmyocardial videointensity using the 10 Hertz frame rate (intermittentimaging) was significantly higher for the PCMB sonicated in the presenceof 100% oxygen.

[0109] Only with the oxygenated PCMB produced a consistent homogenousmyocardial contrast at the doses used for transthoracic imaging. Visualmyocardial contrast was 2+in 20 of the 24 regions following intravenousO₂ PCMB injections compared to 9 or 24 regions following the same doseof RA PCMB (p=0.001). TABLE 4 COMPARISON OF PMVI PRODUCED IN ANTERIORAND POSTERIOR WALL OF LEFT VENTRICULAR SHORT-AXIS VIEW AT MID PAPILLARYMUSCLE LEVEL AFTER INTRAVENOUS VEIN INJECTION OF PCMB SONICATED IN THEPRESENCE OF 100% OXYGEN AND ROOM AIR Microbubble PMVI (units) Conc AntPost Size (μm) (No./hpf) RA PCMB 54 ± 12 19 ± 9  4.0 ± 2.4 109 ± 30 O₂PCMB 70 ± 6* 31 ± 5* 3.9 ± 2.3 108 ± 50

[0110] Interobserver Variability in Microbubble Size. Concentration, andVideointensity Measurements

[0111] Two independent observers measured microbubble size andconcentration of six different slides exposed to either intermittent orconventional ultrasound frame rates. The coefficient of variation formeasurements of microbubble size by two independent observers in sixdifferent samples was 8% (r=0.95; p=0.004), while the coefficient ofvariation for independent measurements of microbubble concentration was9% (r=0.99; p<0.001). The reported mean difference in peak myocardialvideointensity measurements by two independent reviewers fortransthoracic imaging is 4±4 units (r=0.94, SEE=5 units; p<0.001; n=24comparisons), which is well below the 16 unit mean difference inanterior and 13 unit mean difference in posterior peak myocardialvideointensity between O₂ PCMB and RA PCMB. The two investigators werein agreement of the visual degree of contrast enhancement in 37 of the44 regions (84%). Five of the discrepancies were in visual grading of RAPCMB myocardial contrast enhancement (0 vs 1+ in two regions, 1+ vs. 2+in three regions). The three regions where there was disagreement onwhether there was 1+ vs 2+ were assigned a 2+ in the statisticalanalysis.

[0112] Microbubbles containing an albumin shell such as the one used inthis study permit rapid diffusion of soluble gases across theirmembranes. Perfluorocarbon containing microbubbles survive longer thanroom air containing microbubbles with the same membrane because of theslow rate of diffusion of this higher molecular weight gas and itsinsolubility in blood. These microbubbles, however, still contain asignificant quantity of room air gas and thus are not affected by theconcentration gradient that exists across the albumin membrane. Sincesurface tension and absorptive pressures are increased as microbubblediameter decreases, it was hypothesized that the videointensity producedby intravenous PCMB would also be affected by alterations in nitrogenand oxygen concentration inside and outside the microbubble.

[0113] The in vitro studies confirmed that oxygenated blood reduced PCMBsize but did not completely destroy them as has been shown with pureroom air containing albumin microbubbles. Wible J. Jr., et al. (1993),Effects of inspired gas on the efficacy of Albunex® in dogs. Circulation88(suppl):1-401. Abstract. To counter this process, the inventorsattempted to reverse this diffusion gradient by removing nitrogen withinthe microbubbles. It was hypothesized that this would have the oppositeeffect of that seen with oxygenated blood, resulting in nitrogendiffusion inward. The in vitro and in vivo findings of this study appearto support this hypothesis.

[0114] PESDA Microbubble Concentration and Size in Arterial Blood: InVitro Studies

[0115] It has previously been shown that PCMB diameter increases afterinitial exposure to blood at 37° C., most likely from gas expansion fromroom temperature to body temperature. Although this explains whymicrobubble size increased in all samples tested, the PCMB exposed to100% oxygenated arterial blood were significantly smaller in sizecompared to PCMB exposed to room air blood (Table 3). This observationwas seen both following intermittent and conventional imaging. Withoutwishing to be bound by any theory, it is postulated the one potentialexplanation for this is the differences in nitrogen diffusion gradientsacross the microbubble membrane. Since all PCMB in the in vitro studywere sonicated in the presence of room air, there was a significantquantity of nitrogen within the microbubble. Mathematical models havesuggested that microbubbles containing insoluble gases persist longer iftissue and blood contain nitrogen. (Burkard 1994). In the absence ofblood nitrogen (i.e.: 100% oxygenated blood), nitrogen from within thePCMB would have diffused out of the PCMB, reducing their size.

[0116] As expected, microbubble concentrations in room air blood weresignificantly reduced when exposed to higher frame rates. However, thisdestruction by more rapid frame rates was attenuated somewhat when thePCMB were in oxygenated blood. The reason for this difference isunclear. One possibility is that the more rapid diffusion of nitrogenout of the microbubbles in oxygenated blood created a higher internalconcentration of perfluorocarbon, and thus increased the diffusiongradient for the insoluble perfluorocarbon. Due to its low solubility,its enhanced diffusion out of the microbubble would lead to theformation of smaller unencapsulated perfluorocarbon microbubbles. Thehemocytometry resolution would be unable to differentiate encapsulatedfrom unencapsulated microbubbles and thus would count them both. Thisexplanation may also account for the smaller mean microbubble sizeobserved for PCMB exposed to 100% oxygenated arterial blood.

[0117] PCMB Sonicated in a Nitrogen-Free Environment: In VivoDemonstration of Improved Efficacy Over PCMB Sonicated in the Presenceof Room Air

[0118] Based on the in vitro studies, whether the detrimental effects ofa high external oxygen content could be utilized to an advantage bylowering nitrogen content within the PCMB was examined. This wasaccomplished in our study by sonicating the PCMB in the presence of 100%oxygen. Since perfluorocarbons like decafluorobutane act as a mechanicalstabilizer, it was hypothesized that this would create an environmentwhere nitrogen diffuses inward following venous injection, furtherenhancing the stability of the PCMP in blood. This was consistentlyeffective in the closed chest studies in creating greater myocardialcontrast than PCMB sonicated in the presence of room air. Even withintermittent imaging using pulsing intervals as short as 100milliseconds (10 Hertz imaging), visually evident myocardial contrastwas still achieved with the microbubbles sonicated in an oxygen-freeenvironment.

What is claimed is:
 1. A method for delivering a biological agent to specific tissue sites comprising: forming a solution of a plurality of protein encapsulated, insoluble gas-filled microbubbles, said microbubbles conjugated to said biological agent; administering said solution to an animal; so that said protein directs the microbubble-conjugated agent to sites of bioprocessing of said protein and upon dissipation of the microbubble releases said agent.
 2. The method of claim 1 wherein said microbubbles are formed under conditions which lower the partial pressure of nitrogen within the microbubble compared to the partial pressure achieved with room air sonication.
 3. The method of claim 1 wherein said microbubbles are formed in a nitrogen free environment.
 4. The method of claim 3 wherein said environment consists of oxygen.
 5. The method of claim 1 wherein said protein is selected from the group consisting of albumin, human gamma, globulin, human apotransferin, beta lactose and urease.
 6. The method of claim 1 wherein said protein is albumin.
 7. The method of claim 1 wherein said insoluble gas is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and perfluoropentane.
 8. The method of claim 7 wherein said gas is perfluoropropane.
 9. The method of claim 1 wherein said microbubbles are formed by the steps of: mixing an aqueous solution comprising about 2% to about 10% by weight of human serum albumin diluted about two-fold to about eight-fold with 5% to 50% by weight of dextrose; and exposing said solution to a sonication horn in a nitrogen-free environment to create cavitation at particulate sites in said solution thereby generating stable microbubbles from about 0.1 to 10 microns in diameter.
 10. The method of claim 9 wherein said dilution of albumin with dextrose is a three-fold dilution.
 11. The method of claim 9 wherein said human serum albumin is a 5% by weight solution.
 12. The method of claim 9 wherein said dextrose is a 5% by weight solution.
 13. The method of claim 9 wherein said protein is albumin and said biological agent is selected from the group consisting of: an oligonucleotide, a polynucleotide, a ribozyme, naproxen, piroxicam, warfarin, furosemide, phenylbutazone, valproic acid, sulfisoxazole, ceftriaxone, miconazole.
 14. The method of claim 13 wherein said biological agent is an oligonucleotide.
 15. The method of claim 14 wherein said oligonucleotide is a phosphorothioate oligonucleotide.
 16. The method of claim 15 wherein said phosphorothioate oligonucleotide is an antisense oligonucleotide.
 17. The method of claim 9 wherein the solution is exposed to a nitrogen-free environment comprising 100% oxygen.
 18. The method of claim 17 wherein the 100% oxygen is blown into interface between the sonicating horn and the solution.
 19. The method of claim 9 wherein said target site is the liver and the kidney of said animal.
 20. A microbubble composition for ultrasonic imaging or for delivery of a biological agent to a target site comprising: an aqueous suspension comprising a plurality of protein encapsulated insoluble gas-filled microbubbles, wherein the partial pressure of nitrogen in said bubbles is decreased compared to the partial pressure of nitrogen in room air sonicated microbubbles.
 21. The composition of claim 20 wherein said gas-filled microbubbles are nitrogen free.
 22. The composition of claim 20 wherein said protein is selected from the group consisting of albumin, human gamma globulin, human apotransferin, beta lactose and urease.
 23. The composition of claim 20 wherein said protein is albumin.
 24. The composition of claim 20 wherein said gas is a perfluorocarbon gas.
 25. The composition of claim 20 wherein said gas is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and perfluoropentane.
 26. The composition of claim 20 wherein said gas is perfluorobutane.
 27. The composition of claim 20 wherein said gas is perfluoropropane.
 28. The composition of claim 20 wherein said protein is albumin and said biological agent is selected from the group consisting of: an oligonucleotide, a polynucleotide, a ribozyme, naproxen, piroxicam, warfarin, furosemide, phenylbutazone, valproic acid, sulfisoxazole, ceftriaxone, miconazole.
 29. The composition of claim 28 wherein said biological agent is an oligonucleotide.
 30. The composition of claim 22 wherein said gas-filled microbubbles comprise 100% pure oxygen.
 31. A composition for delivery of nucleotide based biological agents to a target site comprising: a plurality of albumin encapsulated insoluble gas-filled microbubbles, wherein the gas filling the microbubbles is nitrogen-free; and a nucleotide based biological agent conjugated to said albumin microbubbles.
 32. The composition of claim 31 wherein said microbubbles are 0.1 to 10 microns in diameter.
 33. The composition of claim 31 wherein said gas is a perfluorocarbon gas.
 34. A method for delivering nucleotide based biological agents to the kidney and liver of animals comprising: forming a solution comprising a plurality of albumin encapsulated insoluble gas-filled microbubbles, said microbubbles conjugated to said nucleotide based biological agent; and administering said solution to an animal; so that said albumin encapsulated microbubble is taken up by said liver and said kidney and upon dissipation of the microbubble, releases said biological agent.
 35. The method of claim 34 wherein said nucleic acid biological agent is selected from the group consisting of an antisense oligonucleotide, antigene oligonucleotide, oligonucleotide probe, or a nucleotide vector.
 36. The method of claim 34 wherein said gas filling said microbubbles is nitrogen free. 